| 作者: |
Qin Jin,Xiaoyu Yang,Shixue Gou,Xiaoyi Liu,Zhenpeng Zhuang,Yanhui Liang, Hui Shi,Jiayuan Huang,Han Wu,Yu Zhao,Zhen Ouyang,Quanjun Zhang,Zhaoming Liu, Fangbing Chen,Weikai Ge, Jingke Xie,Nan Li,Chengdan Lai,Xiaozhu Zhao, Jiaowei Wang,Meng Lia,Lei Li,Longquan Quan, Yinghua Ye, Liangxue Lai,Kepin Wang |
| 摘要: |
Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research. |
